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The evaluation of the radioprotective effect of Triphala (an ayurvedic rejuvenating drug) in the mice exposed to [gamma]-radiation.

Summary

The effect of 0, 5, 6.25, 10, 12.5, 20, 25, 40, 50 and 80 mg/kg b. wt. of aqueous extract of triphala (an Ayurvedic herbal medicine) administrered intraperitoneally was studied on the radiation-induced mortality in mice exposed to 10 Gy of [gamma]-radiation. Treatment of mice with different doses of triphala consecutively for five days before irradiation delayed the onset of mortality and reduced the symptoms of radiation sickness when compared with the non-drug treated irradiated controls. The highest protection against GI (gastrointestinal) death was observed for 12.5 mg/kg triphala, where a highest number of survivors were reported up to 10 days post-irradiation. While 10 mg/kg triphala i.p. provided the best protection as evidenced by the highest number of survivors after 30 days post-irradiation in this group when compared with the other doses of triphala. Toxicity study showed that triphala was non-toxic up to a dose of 240 mg/kg, where no drug-induced mortality was observed. The [LD.sub.50] dose i.p. o f triphala was found to be 280 mg/kg b. wt. Our study demonstrates the ability of triphala as a good radioprotective agent and the optimum protective dose of triphala was 1/28 of its [LD.sub.50] dose.

Key words: Triphala, radiation, mice, survival, acute toxicity, radioprotection, Terminalia chebula Retz., Phyllanthus emblica Linn. or Emblica officinalis Gaertn. and Terminalia bellerica (Gaertn.) Roxb.

Introduction

The search for radioprotectors started with the realization of the need for a safeguard against the military use of atomic weapons. With the recognition that normal tissue protection in radiotherapy is as important as the destruction of the cancer cells, the focus of protection research became more therapy oriented. The use of certain chemical agents may reduce the ill effects of radiation in such conditions. Patt et al. (1949) for the first time observed that the pretreatment of rats and mice with cysteine before exposure to radiation protected them against the radiation-induced sickness and mortality. Subsequently, several chemical compounds were synthesized and tested for their radioprotective ability (Sweeny, 1979). Only sulphydryl compounds have been found to be superior radioprotectors than the other non-sulphydryl compounds but the major drawback of these compounds has been their high toxicity at the optimum protective dose (Sweeny, 1979), which precluded their effective use in men. A turning point cam e with the observation that s-2-(aminopropylamino) ethylphosphorothioic acid (WR-2721) showed substantial and selective protection of normal tissues with little or no protection to the solid tumors (Yuhas, 1980). However, it has also been found to be highly toxic at the optimum protective dose and the possibility of using it on a daily basis was not feasible (Cairnie, 1983). Therefore, it is desirable to search other materials that are less toxic and can offer high protection.

The herbal drugs offer an alternative to the synthetic compounds and have been considered either non-toxic or less toxic and this has given impetus to screen for their radioprotective ability. The mechanism of action of herbal drugs and their extract preparations differ in many respects from that of the synthetic drugs or single substances (Wagner, 1999). It can be characterized as a polyvalent action and interpreted as additive or, in some cases, potentiating. Studies carried out in the past decade and a half have shown that the herbal preparations like Liv. 52, protected mice against the radiation-induced sickness, mortality, dermatitis, spleen injury, liver damage, decrease in the peripheral blood cell counts, prenatal development, lipid peroxidation and radiation-induced chromosome damage (Saini et al., 1984a, b; Saini and Saini, 1985; Saini et al., 1985; Ganapathi and Jagetia, 1995; Jagetia and Ganapathi, 1989, 1991). The brahmarasayana, narasimharasayana, ashwagandharasayana, and amrithaprasham, a group of herbal preparations used to improve the general health, have also been reported to reduce the radiation-induced lipid peroxidation in the liver, and leucopenia in mice (Kumar et al., 1996). Abana, an another herbal preparation, clinically used in India as a cardioprotective agent has also been reported to protect the mice bone marrow against the radiation-induced micronuclei formation (Jagetia and Aruna, 1997).

According to the Ayurvedic system of medicine, the body is composed of tridosha or three humours, vata, translated into wind, corresponds to mind and nervous system, the pitta translated into fire or bile and is responsible for all metabolic transformations including digestion and assimilation of the food, while kapha is translated as water or mucus and it is responsible for the anabolic functions such as development of muscle and bone tissues. Triphala, a compound formulation of the herbs, Terminalia chebula, Phyllanthus emblica or Emblica officinalis and Terminalia bellerica has been described in the Ayurveda as a 'tridoshic rasayan', having balancing and rejuvenating effects on the three constitutional elements that govern human life i.e. vata, pitta and kapha by Charka (1,500B.C.) in the Charaka Samihita (Sharma and Dash, 1998). In the Ayurveda, the word rasayana, is a term used for a therapy that produces sturdiness of the body, the sense organs and the teeth, prevent wrinkles in skin, graying of hair an d promote the immune functions and intellect and render longevity to life (Sharma and Dash, 1998).

Triphala, is one of the important rasayana drugs commonly used in the Ayurvedic system of medicine. This is an antioxidant rich herbal formulation (Vaidya et al., 1998; Jose and Kuttan, 1995; Naiwu et al., 1992, Takagi and Sanashiro, 1996) that has been reported to treat anemia, jaundice, constipation, cough, asthma, fever, eye diseases, chronic ulcers, leucorrhoea, pyorrhea (Nadkarni, 1976) and also assists in the weight loss (Hashimoto and Nakajima, 1997). Triphala and/or its individual plant constituents have been reported to possess anti-bacterial (Nadkarni, 1976; Mehta et al., 1993; Ahmad et al., 1998; Phadke and Kulkarni, 1989; Mehta et al., 1993), antimalarial (Valsaraj et al., 1997), antifungal activity (Dutta et al., 1998; Valsaraj et al., 1997), anti-cancer (Tokura and Kagawa, 1995), anti-mutagenic (Rani et al., 1994; Niwa et al., 1995) anti-allergic (Takagi and Sanashiro, 1996) and anti-viral activities (Valsaraj et al., 1997; Badmaev and Nowakowski, 2000; Yukaka et al., 1996; Kurokawa and Sato, 19 95; Hozumi and Oyama, 1997; El-Mekkawey and Meselhy, 1995) in different study systems. Triphala is a cardio-tonic and exerts its protective effect by improving the blood circulation, reducing the myocardial necrosis (Tariq et al., 1977), serum cholesterol levels and strengthens the capillaries (Tariq et al., 1977; Hussain, 1975; Thakur, 1984; Thakur et al., 1988). It is also hepatoprotective and improves the liver function (Gulati et al., 1995; Anand and Singh, 1997). The decoction of triphala has been found to treat leucorrhea in women (Singh and Londhe 1993). It is an effective laxative and improves the gastrointestinal motility (Tamhane et al., 1997) thereby curing the diseases of gastrointestinal tract (Nadkarni, 1976; Antarkar et al., 1980). Triphala has been reported to possess anti-aging properties and improves the mental faculties (Nadkarni, 1976; Antarkar et al., 1980). Triphala has been found to potentiate the adrenergic function thereby enabling the body to recover from stress. In addition, the imm unomodulatory property (Suresh and Vasudevan, 1994; Rege et al., 1999) may help in increasing the body's defence system resulting in the enhancement of the body resistance against the diseases (Nadkarni, 1976). The diverse medicinal properties attributed to triphala and its antioxidant properties stimulated us to investigate the radioprotective activity of triphala.

The lesson from the experience with radioprotectors world wide is that the animal studies with death as the end point is the most confirmatory, because the 30 days time period after lethal whole body irradiation clearly indicates the capacity of the drug, in test to modulate the recovery and regeneration of the gastrointestinal epithelium and the hemopoietic progenitor cells in the bone marrow, the two most radiosensitive organs that are essential for sustaining of the life. The aim of the present study was to evaluate the radioprotective effect of various doses of triphala in the mice exposed to 10 Gy of whole-body gamma radiation taking survival as the end point.

Materials and methods

The animal care and handling was done according to the guidelines set by the World Health Organization, Geneva, Switzerland and the INSA (Indian National Science Academy, New Delhi, India). Eight to ten week old male Swiss albino mice weighing 30 to 36 g were selected from an inbred colony maintained under the controlled conditions of temperature (23 [+ or -] 2 [degrees]C,) humidity (50 [+ or -] 5%) and light (10 and 14 h of light and dark, respectively). The animals were provided with the sterile food and water ad libitum. Four animals were housed in a polypropylene cage containing sterile paddy husk (procured locally) as bedding throughout the experiment.

Composition of the drug

As the name indicates, triphala (tri = three, phala = fruits) is a mixture of fruits of three plants namely Terminalia chebula Retz. (Family Combretaceae), Terminalia bellerica (Gaertn.) Roxb. (Family Combretaceae) and Phyllanthus emblica Linn or Emblica officinalis Gaertn. (Euphorbiaceae) in powdered form in equal proportions (1:1:1).

Preparation of the extract

The aqueous extract of different batches of triphala powder was prepared as described in the Ayurvedic text. Briefly, 100 grams of the powder (Zandu Pharmaceuticals, India) was boiled in 1000 ml of DDW till the volume was reduced to one fourth of the original (250 ml). The extract was cooled, centrifuged using a cold centrifuge (Sorvall RC-5B, USA) and the supernatant was collected and was concentrated by evaporating its liquid contents. An approximate 26% yield of the extract was obtained.

Preparation of the drug and mode of administration

The required amount of triphala extract (TE) was dissolved in sterile double distilled water (DDW) and administered intraperitoneally.

Determination of acute drug toxicity

The acute toxicity of TE was determined according to Prieur et al (1973) and Ghosh (1984). Briefly, the animals were allowed to fast by withdrawing the food and water for 18 h. The fasted animals were divided into several groups and each group of animals was injected with various doses viz. 200, 220, 240, 260, 280, 300, 350, 400, 450, 500, 750 and 1000 mg/kg body weight (b.wt.) of freshly prepared extract of TE intraperitoneally. Animals were provided with food and water immediately after the drug administration. Mortality of the animals was observed up to 14 days post-drug treatment. Acute [LD.sub.50] of the extract was calculated using a computer program for probit analysis.

Effect of TE on the radiation-induced mortality

The animals were divided into the following groups:

DDW + irradiation group

The animals of this group were administered with 0.01 ml/g b.wt. of sterile double distilled water (DDW) intraperitoneally.

TE + irradiation group

The animals of this group were injected intraperitoneally with 5, 6.25, 10, 12.5, 20, 25, 40, 50 and 80 mg/kg b.wt. of TE consecutively for five days (Jagetia and Aruna 1997).

Irradiation

One hour after the last administration of DDW or TE on the 5th day, the prostrate and immobilized animals (achieved by inserting cotton plugs in the restrainer) of both the groups were whole-body exposed to 0 (Sham-irradiation) or 10 Gy of [blank.sup.60]Co gamma radiation (Gam-matron, Siemens, Germany) in a specially designed well-ventilated acrylic box. A batch of 6 animals was irradiated each time at a dose rate of 1.33 Gy/min at a source to animal distance (midpoint) of 102 cm. The animals were monitored daily up to 30 days post-irradiation for the development of symptoms of radiation sickness, and mortality. The statistical significance between the treatments was determined by "Z" test.

Results

Acute Toxicity

The administration of different doses of TE viz. 200, 220, and 240 mg/kg b.wt. did not induce any mortality during the whole observation period. However, a further increase in the drug dose up to 260 mg/kg b. wt. resulted in a 30% reduction in the survival of mice. An increase in the dose of TE up to 280 mg/kg b. wt. caused a 50% reduction in the survival of mice. 100% mortality was observed at 300 mg/kg and thereafter up to a dose of 1000 mg/kg b. wt. of TE (Table 1).

Effect of TE on the radiation-induced mortality

The mice were treated with different batches of 0, 5, 6.25, 10, 12.5, 20, 25, 40, 50 and 80 mg/kg b.wt. of triphala extract, consecutively for 5 days before whole-body exposure to 10 Gy gamma radiation or not were monitored daily up to 30 days post-irradiation for the development of symptoms of radiation sickness, and mortality. The effect of different doses of TE on the radiation-induced mortality is shown in Table 2 and Fig 1 and the results are expressed as percent survival.

The animals of DDW+irradiation group exhibited signs of radiation sickness within 2-4 days after exposure to 10 Gy of '[gamma]-radiation. The main symptoms included reduction in the food and water intake, irritability, epilation, weight loss, emaciation, lethargy, diarrhea, and ruffling of hairs. Facial edema was also observed in a few animals between one and two weeks after exposure. During the second week after exposure there were a few cases of animals, exhibiting paralysis and difficulty in locomotion. The first mortality in this group was observed on day 4 and 80% of the animals died within 10 days after irradiation, while 96% of the animals died within 30 days of irradiation resulting only in 4% survival by day 30 post-irradiation.

The daily administration of 5, 6.25, 10, 12.5, 20, 25, 40, 50 and 80 mg/kg b.wt. TB for five consecutive days did not cause any drug-induced mortality. The pretreatment of mice with various doses of TE either delayed or reduced the severity of radiation sickness. The onset of radiation-induced mortality was also delayed in TE+irradiation group when compared with the DDW+10 Gy irradiation group. The longest delay was observed for 20 mg/kg TE, where the first mortality was observed by day 10 post-irradiation (Table 2), while a shortest delay was observed for 80 mg/kg, where the first mortality occurred on day 3 post-irradiation.

Treatment of mice with various doses of TE also had an ameliorating effect on the gastrointestinal tract as evidenced by an increase in the 10 day survival of mice, where an increase of 4.58 fold was observed for 12.5 mg/kg, 4.16 fold for 6.25, 10, and 20 mg/kg TE, 3.33 for 25 mg/kg and 2.5 fold for 5 and 40 mg/kg TE (Fig. 1). Majority of the animals (80%) of DDW+ irradiation group, died within 10 days after irradiation, while the TE pre-treatment increased the 10-day survival significantly. A lowest mortality was observed in the animals treated with 12.5 mg/kg TE before irradiation and this decline in mortality was significant (p < 0.00 1). The other doses of TE also reduced the mortality, in comparison with DDW+irradiation, however, a significant elevation in the 10-day survival was observed only for 6.25, 10, 12.5 and 20 mg/kg TE (p < 0.00 1) treatment. 5 and 25 mg/kg of TE also protected mice against the radiation-induced mortality, however, the differences were statistically non-significant.

Analysis of thirty day survival revealed a drug dose dependent increase in the survival of irradiated mice up to a dose of 10 mg/kg in the TE+irradiation group, where a highest survival of 58.33% was observed as compared to the DDW+irradiation, where only 4% animals survived at the end of 30 days (Table 2). A further increase in the drug dose to 12.5 mg/kg resulted in 8.33% reduction in the survival, when compared with the 10 mg/kg TE. Increase in TE doses further, resulted in a consistent decline in the animal survival reaching a nadir at 50 mg and 80 mg/kg, where no survivors could be reported at the end of 30 days (Fig 1). TE administration before irradiation increased the survival significantly for 5 to 25 mg/kg (p < 0.02 to 0.00 1). However, the number of survivors was highest (58.33%) after pretreatment of mice with 10 mg/kg of TE, and hence it was considered the optimum dose for radioprotection. The optimum radioprotective dose of 10 mg/kg TE was found to be 1/28 of the [LD.sub.50] dose (280 mg/kg b.wt.), which was far below the [LD.sub.50] dose.

Discussion

Ayurveda (in Sanskrit Ayu = life and veda = knowledge), the Indian system of medicine, dating back to 5000 years has been an integral part of Indian culture and materia medica. The Ayurveda, extensively uses the plant-derived compound formulations for the treatment of various ailments after a careful study into the type of the disease (Sivarajan and Balachandra, 1996). Often the drugs formulated are such that they have the desired activity with the adequate potency and are devoid of untoward side effects. As it is observed that the desired activity is rarely present in adequate potency in a single plant and it may also contain unwanted activities. Therefore, several plants with the common desired activities and varied undesirable activities are selected so that the final formulation will have a concentrated desired activity and the undesired activities will be absent or diluted. Further, it is also observed that in such formulation, certain other compound may be of help in enhancing of the potency of the acti ve compounds resulting in an additive or synergistic positive effect, which may be of immense benefit to the patient (Kulkarni, 1997). Keeping this Ayurveda philosophy in mind, triphala, an herbal rasayans preparation, credited with diverse beneficial properties like anti-aging, antimutagenic, anticancer, antibacterial, anti-viral, cardioprotective, hepatoprotective, anti-stress, cleanser of colon, gas distentioner, antidiabetic, antiparasitic, anti-diarrhea and antianemic (Nadkami, 1976; Mehta et al., 1993; Ahmad et al., 1998; Phadke and Kulkarni, 1989; Niwa et al., 1995; Valsaraj et al., 1997) has been selected for the evaluation of its radioprotective activity in mice.

The animals of the irradiated group (DDW + irradiation) exhibited signs of radiation sickness within 2-4 days after exposure to 10 Gy and the symptoms included reduction in the food and water intake, irritability, epilation, weight loss, emaciation, lethargy, diarrhea, and ruffling of hairs. The death of 80% of the animals exposed to 10 Gy of radiation within 10 days is because of functional failure of the gastrointestinal tract (Bond et al., 1965; Uma Devi et al., 1999). The remaining 16% animals died within the next 20 days exhibiting hemopoietic syndrome and the charecteristic symptoms like, irritability, epilation, weight loss, emaciation, lethargy and ruffling of hairs (Bond et al., 1965; Uma Devi et al., 1999). It is a well-established fact that ionizing radiation at cellular level can induce damage in the biologically important macromolecules such as DNA, proteins, lipids and carbohydrates in the various organs. While some damage is expressed early others are expressed over a period of time depending u pon the cell kinetics and the radiation tolerance of the tissues. Like in chemotherapy, the effect of whole body irradiation is mainly felt by the highly proliferating germinal epithelium, gastrointestinal epithelium and the bone marrow progenitor cells. Of these the germinal epithelium does not have a life supporting function to the exposed individual, while the gastrointestinal epithelium and the bone marrow progenitor cells are crucial for sustaining of life and any damage to these cells will impair the normal physiological processes drastically. The gastrointestinal epithelium is less sensitive than the bone marrow progenitor cells but as the cell transit time is quick, it is expressed earlier than the hemopoetic syndrome (Bond et al., 1965). In mice death within 10 days post-irradiation is due to the gastrointestinal damage (Uma Devi et al., 1999). The bone marrow stem cells are more sensitive to radiation damage than the intestinal crypt but, the peripheral blood cells have a longer transit time than th e intestinal cells and hence the gastrointestinal syndrome appears earlier than the bone marrow syndrome and in mice death due to irradiation from 11 to 30 days is due to the hemopoetic damage inflicted by radiation (Bond et al., 1965; Uma Devi et al., 1999).

The pretreatment of mice with different doses of TE resulted in a dose dependent reduction in the radiation-induced mortality up to 10 mg/kg and a further increase in the drug dose resulted in the decline in the animal survival when compared with the 10 mg/kg TB. The earlier studies on radioprotection have shown that an agent in test (for radioprotective action) acts only at a particular dose range and above which it may not be protective and some times can even be toxic (Thomson, 1962; Yuhas and Storrer, 1969). The active principle of Plumbago rosea, the plumbagin at pico to femto gram range has been reported to stimulate the granulocytes in vitro, while at higher doses it had immunosuppressive activity (Wagner et al., 1988). The reason may be that after a particular concentration, a compound instead of being an anti-oxidant may act like a pro-oxidant inducing toxic symptoms resulting in the death. This is the reason that TE has optimum protection at 10 mg/kg and the higher doses result in the decline in the protective action of TE. The TE pretreatment provided protection against the radiation sickness and mitigated the animal sufferings. Reports regarding the use of TE to protect against the radiation damage are unavailable, as this is probably the first report regarding the radioprotective action of TE. However, certain other herbal preparations like Liv. 52, and abana have been reported to protect the mice against the radiation-induced sickness, mortality, dermatitis, spleen injury (Saini et al., 1984 a, b) and radiation-induced chromosome damage (Jagetia and Ganapathi, 1989, 1991; Jagetia and Aruna, 1997). The brahmarasayana, narasimharasayana, ashwagandha-rasayana, and amrithaprasham, a group of herbal preparations used to improve the general health and debility, have been reported to reduce the radiation-induced lipid peroxidation in the liver, and leucopenia in mice (Kumar et al., 1996).

The pattern of survival in TE+irradiation group was similar to that of the irradiated control group except that the mortality was delayed. This clearly indicates the effectiveness of TE in arresting GI death, where the number of survivors for 5, 6.25, 10, 12.5, 20, 25 and 40 mg/kg was higher than that of the irradiated control. The reduction in GI death may be due to the protection of intestinal epithelium, which would have allowed proper absorption of the nutrients. Triphala has been used as laxative to support the body's vitality in man and it even stops diarrhea. Our findings support the contention that triphala may protect the gastrointestinal tract epithelium against the toxic insult of radiation, protecting against the GI death in this study. It has been reported that, Terminalia chebula, an important constituent of Triphala, mitigated the cysteamine-induced duodenal ulcers in rats by increasing the beta-glucuronidase activity in the Brunner's glands (Nadar and Pillai, 1989) and protected the epithelial cells against the cytopathic effects caused by influenza A virus (Badmaev et al., 2000). Another herbal drug Liv. 52 has been reported to protect the intestinal epithelium against the radiation-induced damage (Saxena and Goyal, 1998).

The pretreatment of mice with TE significantly reduced the bone marrow deaths in the TE+irradiation group, especially at a dose of 5 to 25 mg/kg, where a significant elevation in the survival has been observed. This increase in the 30 day survival may be owing to the protection afforded by TE to the bone marrow stem cell compartment, which continued to supply the requisite number of cells in the survivors. The bone marrow cells have been reported to be protected against the radiation-induced damage by various other plant formulations (Saini et al., 1984a, b; Jagetia and Ganapathi, 1989, 1991; Jagetia and Aruna, 1997; Kumar et al., 1996). Further, triphala, and its constituents are reported to possess antimicrobial activity (Mehta et al., 1993; Dutta et al., 1998; Ahmad et al., 1998; Phadke and Kulkarni, 1989; Valsaraj et al., 1997), which would have also been responsible for the radioprotective action of TE. One of the constituents of triphala, the Phyllanthus emblica, has been found to be immunomodulator (Su resh and Vasudevan, 1994; Rege et al., 1999) and this would have increased the body's defence system by increasing the immunity. Further the antimicrobial action of triphala would have prevented the localization of the pathogenic microbes in the GI tract and bacterial infection, resulting in the observed radioprotection.

TE is mainly composed of Terminalia chebula, Phyllanthus emblica and Terminalia bellerica in equal proportions and each plant has been utilized to treat various ailments and diseases in the Ayurvedic system of medicine. Terminalia chebula is a commonly advocated agent in Ayurveda for improving gastrointestinal motility (Tamhane et al., 1997). The water and chloroform extracts of it have been shown to protect against the sodium azide and 4-nitro-o-phenylenediamine induced mutagenesis (Grover and Bala, 1992). Recently, it has also been reported to possess antioxidant activity and prevent the TPA-induced DNA breaks in human white cells (Naiwu et al., 1992). Similarly, Terminalia bellerica has been found to contain anti-HIV-1, antimalarial, and antifungal activity (Valsaraj et al., 1997). The alcoholic extract of this plant reduced the serum GOT, GPT and LDH activity, caused a significant reduction in fatty acid levels, and protected against the myocardial necrosis (Tariq et al., 1977). Phyllanthus embelica has a lso been found to be rich in ascorbic acid contents and ascorbic acid has already been reported to reduce the radiation-induced sickness and mortality (Redpath et al., 1982) and to protect mice bone marrow cells against the radiation-induced chromosome damage (Sarma and Kesavan, 1993). In addition to ascorbic acid, the components of triphala, Phyllanthus emblica, Terminalia chebula and Terminalia bellerica also contain ellagic acid, which has been reported to decrease the bone marrow micronuclei formation in the mice (Thresiamma et al., 1996). Phyllanthus emblica has also been reported to contain flavonoids (Jose and Kuttan, 1995), a class of compounds reported to be possess antioxidant and free radical scavenging activities (Tanaka, 1994; Uddin and Ahmad, 1995; Korina and Afanasev, 1997; UmaDevi et al., 2000). Certain flavanoids have been found to protect against the radiation-induced DNA damage (Shimoi et al., 1994, 1996, 1997; Uma Devi et al., 1998) and mortality (Uma Devi et al., 1999). The aqueous, aceto ne and chloroform extracts of Emblica officinale found to have antimutagenic effect (Grover and Kaur, 1989).

The exact mechanism of action of TE is not known, however, it may scavenge free radicals produced by radiation and thus reduce the radiation-induced damage to the cellular DNA. The presence of ascorbic acid and the flavonoids like quercetin may be responsible for this action as these compounds are reported to protect the DNA from radiation-induced micronuclei in mice (Sarma and Kesavan, 1993; Shimoi et al., 1997). While testing NO (nitric oxide) scavenging activity of several agents, triphala was found to scavenge the nitric oxide production in vitro (unpublished data). The aqueous extract of one of the constituents of TE, Phyllanthus emblica has been reported to be a potent inhibitor of lipid peroxidation formation, and scavenger of hydroxyl and superoxide radicals in vitro (Jose and Kuttan, 1995). Photochemical studies have shown that Terminalia bellerica contains bellericanin, ellagic acid, gallic acid, chebulagic acid, ethyl gallate and [beta]-sitosterol. Terminalia chebula has been found to contain chebu lin, terchebin, chebulagic acid, chebulinic acid, corilagin, ellagitannin, ellagic acid, gallic acid, [beta]-D-glcogallin, and terchebin. The Phyllanthus emblica has been reported to be a rich source of vitamin C and also contains terchebin, corilagin, tannins, ellagic acid, phyllembic acid, gallic acid and flavonoids in different proportions depending on the season, type of climate and the plant processing (Chemexcil, 1992; Satyavati et al., 1987; Wealth of India 1952, 1976; Rastogi and Mehrotra, 1990; Jose and Kuttan, 1995). Most of these compounds have been reported to possess antioxidant and free radical scavenging activities (Tanaka, 1994; Uddin and Ahmad, 1995; Korina and Afanasev, 1997) and increase the antioxidant enzymes (Kong Ah-Ng et al., 2000). The presence of various antioxidant compounds in triphala might have been responsible for the observed radioprotection by scavenging of free radicals generated by radiation exposure. Alternatively triphala might have increased the intracellular level of GSH , and stimulated the immune systems which could have provided protection against the radiation-induced mortality.

Conclusions

From our study it is clear that TE, a plant based formulation provided protection against the radiation-induced sickness and mortality and the optimum protective dose of 10 mg/kg i.p. is far below the [LD.sub.50] (280 mg/kg) dose. The exact mechanism of action of TE is not known, however, it may scavenge free radicals produced by radiation and thus inhibit radiation-induced damage to the cellular DNA. We have observed scavenging of NO (nitric oxide) radicals in vitro by TE (unpublished data) and this testifies to our belief that one of the mechanisms of radioprotection by triphala may be owing to the scavenging of free radicals generated by radiation exposure. Alternatively, it may also increase GSH levels and may reduce the radiation-induced lipid peroxidation. Since significant protection is obtained at a very low non-toxic dose the extract may have an advantage over the known radio-protectors available so far. Studies are planned to explore the applicability of triphala in cancer treatment by looking for t he preferential protection to the normal tissues and its clinical applicability for cancer cure in the fractionation regime. Since this formulation has been in use in India for the last 5000 years, the clinical acceptability shall not be a problem.

[FIGURE 1 OMITTED]
Table 1

Acute toxicity of the extract of triphala on the survival of mice.

Triphala Mortality on different days post drug treatment
mg/kg
 1 2 3 4 5 6

 200
 220
 240
 260 1 2
 280 1 2 1
 300 3 4 3
 350 6 2 2
 400 7 3
 500 9 1
 750 10
1000 10

Triphala Mortality on different days post drug treatment
mg/kg
 7 8 9 10 11 12

 200
 220
 240
 260
 280 1
 300
 350
 400
 500
 750
1000

Triphala Mortality on
 different days post
 drug treatment
mg/kg
 13 14 % Survivors Survivors/Total

 200 100 10/10
 220 100 10/10
 240 100 10/10
 260 70 7/10
 280 50 5/10
 300 0 10/10
 350 0 10/10
 400 0 10/10
 500 0 10/10
 750 0 10/10
1000 0 10/10
Table 2

Effect of various doses of triphala on the survival of mice exposed to
10 Gy of [gamma]-irradiation.

Triphala Mortality on different post-irradiation days
(mg/kg)
 1 2 3 4 5 6

 0 - - - 3 3 4
 5 - - - - - -
 6.25 - - - - - -
10 - - - - - -
12.5 - - - - - -
20 - - - - - -
25 - - - - - -
40 - - - - - 1
50 - - - 2 1 1
80 - - 5 5 - 1

Triphala Mortality on different post-irradiation days
(mg/kg)
 7 8 9 10 11 12

 0 1 2 2 5 1 1
 5 - 2 2 2 - 1
 6.25 - 1 - 1 - 1
10 - - 2 - - -
12.5 - - 1 - 1 -
20 - - - 2 - 1
25 - - 2 2 - 1
40 2 1 1 1 1 -
50 2 2 3 1 - -
80 - - 1 - - -

Triphala Mortality on different post-irradiation days
(mg/kg)
 13 14 15 16 17 18

 0 - 1 - - 1 -
 5 - 1 - - - -
 6.25 - - - - - 1
10 - 1 1 - - -
12.5 1 - - - - 1
20 1 - - 1 1 -
25 - 1 - - - -
40 - 1 1 - - -
50 - - - - - -
80 - - - - - -

Triphala Mortality on different post-irradiation days
(mg/kg)
 19 20 21 22 23 24

 0 - - - - - -
 5 - - - - - -
 6.25 2 - - - - -
10 - - - - - -
12.5 - - - - - -
20 1 - - - - 1
25 - - - - - -
40 - - - - - -
50 - - - - - -
80 - - - - - -

Triphala Mortality on different post-irradiation days
(mg/kg)
 25 26 27 28 29 30

 0 - - - - - -
 5 - - - - - -
 6.25 1 - - - - -
10 - 1 - - - -
12.5 1 - - 1 - -
20 - - - - - -
25 - - - - - -
40 - - - - - -
50 - - - - - -
80 - - - - - -

Triphala No. of Total
(mg/kg) Survivors


 0 1 25
 5 4 (a) 12
 6.25 5 (b) 12
10 7 (c) 12
12.5 6 (c) 12
20 4 (a) 12
25 4 (a) 12
40 3 12
50 0 12
80 0 12

- P < (a)= 0.02

(b)= 0.01 and

(c)= 0.001


Acknowledgements

We thank Prof. M. S. Vidyasagar, and Dr. J. Velumurugan, Department of Radiotherapy and Oncology, Kasturba Medical College, Manipal, India for providing the necessary irradiation facilities and help in radiation dosimetry.

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G. C. Jagetia (1), M. S. Baliga (1), K. J. Malagi (2) and M. Sethukumar Kamath (2)

(1) Department of Radiobiology, Kasturba Medical College, Manipal, India

(2) Department of Ayurveda, Kasturba Hospital, Manipal, India

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Dr. Ganesh Chandra Jagetia, Department of Radiobiology, Kasturba Medical College, Manipal 576 119, India.

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Author:Jagetia, G.C.; Baliga, M.S.; Malagi, K.J.; Kamath, M. Sethukumar
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
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Date:Mar 1, 2002
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